Project Abstract The majority of breast cancers are estrogen receptor positive (ER+) and ER+ breast cancer is the leading cause of breast cancer mortality. Despite the widespread adoption of adjuvant endocrine therapy for patients with ER+ breast cancer, almost all patients with ER+ metastatic disease will eventually develop resistance to endocrine treatments. A number of mechanisms of endocrine resistance were proposed but for the most these were predominantly based on preclinical evidence. Recently, our group and a number of other groups identified estrogen receptor (ER) ligand-binding domain (LBD) mutations as the most prevalent genomic mechanism of endocrine resistance identified in patients with ER+ metastatic breast cancer. These mutations are associated with decreased overall survival in ER+ metastatic breast cancer. The addition of CDK4/6 inhibitors (CDK4/6i) to endocrine treatment has been shown to improve progression free survival in patients with metastatic ER+ breast cancer, but have not been shown to improve overall survival. Despite this advance, patients with metastatic disease invariably develop resistance to this combined therapy, exhaust other chemotherapy options, and ultimately die of their disease. There is an urgent need to develop new therapeutic strategies to overcome resistance to endocrine treatment and CDK4/6i. Our preliminary data suggests that selective inhibition of CDK7 can overcome resistance to endocrine resistance driven by the ER mutations and resistance to CDK4/6i. CDK7 has key roles in transcriptional and cell cycle regulation. SY1365 is a selective CDK7 inhibitor that is in early stages of clinical development for cancer treatment. This study is designed to comprehensively study the activity of SY1365 as a single agent and in combination with fulvestrant for the treatment of ER mutant endocrine resistant and CDK4/6i resistant ER+ metastatic breast cancer. We will investigate the mechanisms by which CDK7 inhibition exerts anti-tumor activity and overcomes resistance to endocrine treatment driven by the ER mutations and resistance to CDK4/6i. Because CDK7 phosphorylates RNA polymerase II and also functions as a CDK-activating kinase, we will test the effects of SY1365 on nascent RNA and mRNA transcripts, CDK2 phosphorylation and cell cycle progression in ER+ breast cancer models. We will study mechanisms of resistance to CDK7 inhibition and synthetic lethal vulnerabilities in the presence of SY1365. Lastly, we will test the activity of SY1365 in endocrine resistant and CDK4/6i resistant PDX models derived from metastatic tumors from patients with ER+ disease. Results from this study have the potential to lead to a clinical trial testing the efficacy and on target activity of a new class of drugs, selective CDK7 inhibitors, for the treatment of endocrine and CDK4/6i resistant ER+ metastatic breast cancer. These results could lead to a new therapeutic strategy to improve outcomes in a substantial number of patients with metastatic ER+ breast cancer.